Biology Reference
In-Depth Information
( Cheung et al., 2006; Torrecilla et al.,2000 ), obelin ( Stepanyuk et al., 2005 ),
mitrocomin ( Inouye and Sahara, 2009 ), clytin ( Inouye, 2008 ), and photina
( Bovolenta et al., 2007 ). In addition, there is a growing number of aequorin-
derived bioluminescence resonance energy transfer (BRET)-based complexes
such as the GFP-aequorins ( Ashworth and Brennan, 2005; Baubet et al.,2000;
Martin et al.,2007;Rogerset al., 2005, 2007 ), as well as other wavelength-shifted
variants ( Gorokhovatsky et al.,2004 ). To date, apoaequorin alone, or apoae-
quorin in tandem with another BRET protein, has been genetically expressed in a
diverse range of di
erent species; either in the whole organism or in specific
tissues within an intact organism (See Table I and Fig. 1 ). For example, apoae-
quorin has been ubiquitously expressed in whole zebrafish embryos ( Cheung
et al., 2006 ) or specifically targeted to the Malpighian tubules in Drosophila
( Rosay et al., 1997 ), while the BRET complexes GFP-apoaequorin and YFP-
apoaequorin have been specifically targeted to neuronal cell subsets of Drosophi-
la ( Martin et al., 2007 ), and the endodermis and pericycle of Arabidopsis roots
( Kiegle et al., 2000 ), respectively. Furthermore, apoaequorin or apoaequorin-
BRET complexes have been expressed either ubiquitously in the cytosol of cells in
culture or, using specific targeting sequences, in distinct organelles of cells in
culture (see Table II , and a recent review by Gerasimenko and Tepikin, 2005 ).
Specific organelles targeted include: the ER ( Montero et al.,1997 ), mitochondria
( Rizzuto et al.,1992 ), the Golgi apparatus ( Pinton et al., 1998 ), the nucleus ( Brini
et al., 1993, 1994 ), gap junctions ( George et al.,1998 ), subplasma membrane
domains ( Marsault et al., 1997; Nakahashi et al., 1997 ), secretory vesicles
( Mitchell et al., 2001 ), and the outer mantle of secretory granules ( Pouli et al.,
1998 ), to name but a few examples.
In this chapter, we focus on describing the practical uses of bioluminescent GET-
CRs, especially those based on apoaequorin. In addition, we provide the reader (in
table form) with a review of the literature to date listing representative examples of
whole organisms, tissues, and cells that have been transfected with apoaequorin or
an apoaequorin-BRET complex, as well as a list of organelles and subcellular
domains that have been specifically targeted (see Tables I and II ). We also summa-
rize di
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erent strategies used for loading various derivatives of the apoaequorin
cofactor, coelenterazine ( Shimomura et al.,1989 ) into cells, tissues, and intact
organisms (summarized in Table III ). Furthermore, we describe recent advances in
detection and imaging technologies used to measure and visualize light generated by
the aequorin-Ca 2 þ luminescent reaction within cells, tissues, and intact organisms
(summarized in Table IV and illustrated in Figs. 2 and 3 ). Our hope is that this
chapter will provide a starting point for researchers wishing to use GET-CRs to
measure or visualize Ca 2 þ dynamics from cells, tissues, or intact organisms. Fur-
thermore, the references provided in Tables I-IV should lead them to more detailed
information regarding a biological system and/or experimental setup that will com-
plement their own research interest. For loading holoaequorin into cells and embry-
os, we refer readers to the practical methodologies described in Miller et al. (1994) ,as
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